Hi. I am doing an agarose gel electrophoresis. After running PCR samples in a 2% agarose gel at 90V x 45' I obtained the results in the image.
The ladder bands are not properly stained. For example, the bands at the bottom have more intensity than the reference (500bp). Using another DNA marker I got similar results.
I am using a recent GelRed added during the gel preparation at 0.1X concentration (which worked fine before). Increasing the concentration to 1X resulted in all ladder bands being very bright. The DNA marker used is brand new. The TBE used was new.
Why are the bands not staining properly? Thanks.
The only reason to worry about the seemingly too high intensity of the low Mw bands is if you want to quantify the amount of DNA present in each band. I suspect that the difference with the pattern on the right described by the manufacturer has to do with the fact that you stain with GelRed, which is more sensitive than ethidium bromide, which was probably used for staining the sample on the right. If you wish to use the marker ladder to quantify your bands, I would suggest that you try ethidium bromide for staining or maybe 1 x GelRed, but reducing the amount of ladder and sample loaded. Ethidium bromide is widely ddescribed as a dangerous mutagen and carcinogen, but don't forget that GelRed, GelGreen, SybrGreen I and II are all intercalatig agent and may not be as safe as touted by the manufacturers.
GelRed is known to be highly sensitive and would tend to give you brighter bands in your ladder. EtBr is the alternative you can try. Dyes such as GelRed have been studied to show shift and smearing of the sample bands. Quantitation could be performed using Nanodrop Spectrophotometer or Quantus Fluorometer to obtain precise results.
1. Which DNA Ladder do you use? Is that the one you presented here on right (GeneRuler 100bp)?
2. What is the content of the loading dye you use for your DNA samples?
Thanks for the answers. I understand that if my goal is not to quantify DNA, I should not be worried about the intensities of the bands. However, I still would like to correct that issue in case I need a good photo of the gel. I will try to use EtBr as suggested by Pierre and Joyleen.
Saber Delpasand Khabbazi 1. I am using GeneRuler100 bp. I also tried with 100bp Plus Opti-DNA Marker, obtaining similar results.
2. I use 1ul LD + 5ul DNA sample. For the DNA marker, I use 1ul LD + 3ul H2O + 2ul DNA Marker.
Have you encountered something similar during your experiments?
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